Flaviviruses represent serious global health challenges. Research on these viruses, including Zika virus (ZIKV), is crucial to help prevent the spread of epidemics and will ideally result in the availability of therapies. Antiviral development would be aided by a deeper understanding of the mechanisms of viral replication. The flavivirus NS5 protein encodes multiple critical proviral activities. These include the enzymatic methyltransferase and polymerase activities, which are required for viral genome amplification inside infected cells. We have also identified an NS5 activity that is required for the spread of virus between cells, presumably at the level of infectious virus assembly or release. Finally, NS5 is also a major interferon antagonist that permits infection in the face of potential host innate immune responses. Apart from the basic elements of the methyltransferase and polymerase enzymatic activities, such as substrate binding and active site residues, little to nothing is known about the sequence determinants that contribute to this vast array of activities. We hypothesize that ZIKV NS5 encodes numerous overlapping functions that can be genetically separated. To identify these determinants, we will generate libraries comprised of all possible single amino variants in the NS5 protein. To do so, we will use a novel infectious clone of a contemporary ZIKV strain that we generated. We will screen this library for the capacity to undergo RNA replication and spread by infecting cells and looking at which viruses are present after only one day (to allow only replication, not spread, to occur) and several days (which will allow spread of viruses between cells). The mutational tolerance for each activity will be determined by deep sequencing, the viral RNAs in infected cells at the two time points and comparing them to the initial plasmid library. We will also passage this library in cells treated with interferon to select mutants that retain the capacity to inhibit STAT2, a mediator of innate immune response. This activity will be similarly defined by deep sequencing. The sequencing approach will use powerful next-generation sequencing technologies to assay all possible NS5 single amino acid mutations simultaneously and is a highly scalable strategy to study ZIKV evolutionary potential. The results of this project will provide in-depth insights into flavivirus host cell interactions and replication mechanisms that may aid in the development of therapeutic efforts for ZIKV and could perhaps be further applied in combating other future virus outbreaks.